Project Description The broad objective of this proposal is to understand the mechanism of homologous recombination and its relationship to chromosome maintenance in both Bacteria and Eukarya. Our approach is to reconstitute increasingly more complex reactions that recapitulate steps of the recombination process, using purified proteins from Escherichia coli and from Saccharomyces cerevisiae, and to use sophisticated biochemical and biophysical methods of analysis, which include single-molecule imaging. The specific aims of this proposal are divided along two related aims. The first set of aims describes our ongoing efforts to mechanistically understand the steps of E. coli recombination in vitro. This aim includes studies on the function of RecN protein, and the further elaboration of mechanism of the DNA homology search by RecA. The second major aim is to continue to biochemically reconstitute the processes that comprise recombination in S. cerevisiae. This aim includes studies of the coupling of DNA resection to DNA pairing, and the mechanism of the DNA homology search by Rad51.